INSTRUMENTAL METHODS OF

ANALYSIS
ASSIGNMENT 1 & 2

-SANJHI NEHROTRA (BCT 2ND YEAR)

 Q1. Explain the different types of paper chromatography and their
applications in cosmetics.

ANS. Paper chromatography was used to separate the mixture of organic

substances such as dyes and amino acids only. But now this method has been
perfected to separate cations and anions of inorganic substances as well. This
technique is a type of partition chromatography in which the substances are
distributed between two liquids, i.e., one is the stationary liquid (usually water)
which is held in the fibers of the paper and called the stationary phase; the other is
the moving liquid or developing solvent and called the moving phase. The
components of the mixture to be separated migrate at different rates and appear as
spots at different points on the paper.

Types of Paper Chromatography

(a) Descending Chromatography. When the development of the paper is done by

allowing the solvent to travel down the paper, it is known as descending technique.
The apparatus consists of a well-sealed glass tank of suitable size and shape which
is provided with a trough for the mobile phase in the upper portion. The paper with
the sample spotted is inserted with the upper end in the trough containing the
mobile phase, the jar itself having been equilibrated with the mobile phase prior to
elution.

The advantage of the descending technique is that the development can be

continued indefinitely though the solvents run off at the other end of the paper.

(b) Ascending Chromatography. When the development of the paper is done by

allowing the so to travel up the paper, it is known as ascending technique.

In ascending chromatography, the mobile phase is placed in a suitable container at

the bottom of the chamber or in the chamber itself. The samples are applied a few
centimeters from the edge of the paper suspended from a hook. Alternatively, the
paper may be rolled into a cylinder, held together by staples, strings or plastic clips.

(c) Ascending-Descending Chromatography. It is a hybrid of the above two

techniques. In this technique, the upper part of the ascending chromatography can
be folded over a glass rod allowing the ascending development to change over into
the descending after crossing the glass rod.

(d) Radial Paper Chromatography. This is also known as circular paper

(chromatography). This makes use of radial development. In this technique, a
circular filter paper is employed. Then the various material to be analyzed is placed
at the center. After drying the spot the paper is fixed horizontally on the petri-dish
possessing the solvent so that the tongue or the wick of the paper dips into the
solvent. Cover the paper by means of a petri-dish cover. The solvent rises through
the tongue or the wick. When the solvent front has moved through a sufficiently
large distance, the components get separated in the form of concentric circular
zones.

(e) Two dimensional chromatography. In this, a square or rectangular paper is

used. The sample is applied to one of the corners. The second development is
performed at right angle to the direction of the first run. This type of
chromatography can be carried out with identical solvent systems in both the
directions or by two solvent systems.

This technique can be understood by spotting the sample at the lower corner of a
rectangular sheet of filter paper so that the spot is situated above the surface of the
developing solvent in the trough. The dried paper is then kept with its edge in the
solvent and developed by either ascending or descending technique. When the
solvent reaches the opposite edge of the paper, it is removed from the tank and
dried! The solvent system is now changed to a second liquid. The filter paper is now
rotated through 90° so that the edge having the series of spots is now at the bottom,
just above the solvent trough and the chromatogram is run as before.

APPLICATIONS OF PAPER CHROMATOGRAPHY :

• Reaction monitoring. Over a period of time, the concentration of reactants

decreases, whereas the concentration of production increases in a chemical reaction.
One can get a fair idea of the progress of the reaction by spotting the reactants and
developing the chromatogram over different time intervals. The availability of
densitometers made quantitative estimations possible, but traditionally the technique
was used for qualitative monitoring. However, as a reaction monitoring option, the
rapid methods using spectroscopic techniques are limiting the paper chromatography
application.
• To ensure the control of the purity of pharmaceuticals.
• For the study of ripening and fermentation.
• For the analysis of the reaction mix in biochemical laboratories. Certain
organic compounds such as carbohydrates and amino acids are identified or detected
from a complex mixture of organic compounds with the help of paper chromatography.
It is useful in the separation of anions and amino acids.
• To detect contaminated substances in beverages and foodstuffs. Both natural
and synthetic food colors are added to foods to improve their acceptability and to make
them more popular. Paper chromatography has been primarily used for analysis of food
colors in ice creams, sweets, drinks and beverages, jams and jellies. To ensure that no
non-permitted coloring agents are added to the foods, only edible colors are permitted
for use. That's how quantification and identification becomes more important.
• For the analysis of cosmetics.
• Separation and purification techniques for components. or components of
mixture, paper chromatography has been put to use as a purification and isolation
technique. Using spectro-photometric methods, the separated components on the paper
are cut, dissolved in suitable solvents and their absorption is characterized at specific
wavelengths.
• Forensic testing. For investigation of crimes, paper chromatography is useful in the
field of forensic science, as this process can be successfully carried out with even very
small quantities of material. Using this technique, samples from crime scenes are
collected to be analyzed and identified. Used in DNA and RNA fingerprinting. Moreover,
to detect the presence of alcohol or chemicals in blood, pathological laboratories use
paper chromatography.
• Performance-enhancing drug testing.
• Qualitative analysis. To analyze or separate the different constituents of a mixture,
paper chromatography is used. It is one of the methods of qualitative analysis. We can
say it as a useful tool for separating polar as well as non polar solutes. To analyze the
different compounds in drugs, most of the pharmaceutical companies use this
technique.
It is used in determining the pollutants in water and testing of antibiotics.

Q1. Explain the different types of column chromatography and mention

the different parts of the coloumn.
ANS. Column chromatography is described as the useful technique in which the
substances to be isolated are presented onto the highest point of a column loaded with an
adsorbent (stationary phase), go through the column at various rates that rely upon the
affinity of every substance for the adsorbent and the solvent or solvent mixture, and are
typically gathered in solution as they pass from the column at various time. The two most
common examples of stationary phases for column chromatography are silica gel and
alumina while organic solvents are regarded as the most common mobile phases.

The main principle involved in column chromatography is the adsorption of the solutes of
the solution with the help of a stationary phase and afterward separates the mixture into
independent components. At the point when the mobile phase together with the mixture
that requires to be isolated is brought in from the top of the column, the movement of the
individual components of the mixture is at various rates.
The components with lower adsorption and affinity to the stationary phase head out
quicker when contrasted with the greater adsorption and affinity with the stationary
phase. The components that move rapidly are taken out first through the components that
move slowly are eluted out last. The adsorption of solute molecules to the column happens
reversibly.

Types of coloumn chromatography

Adsorption Chromatography
It is a type of liquid chromatography which retains the compounds from the mixture,
adsorbed on the surface of the stationary phase.
 Usually, the forces involved in binding the compound with the solid support are Van
Der Waals forces and steric interactions. Besides, adsorption chromatography refers to
as solid-liquid chromatography. This chromatography was first invented by the Russian
botanist Mikhail Tsvet in 1901

The invention of adsorption chromatography is associated with its very first example.
The chlorophyll and carotenoid pigments were formerly separated using calcium
carbonate, sucrose, and alumina as a stationary phase, with petroleum and
ether/ethanol mixtures as eluents.

Ion Exchange Chromatography

Ion chromatography or ion-exchange chromatography is another type of column

chromatography that mostly separates polar and ionic compounds. The basic
principle lies in the molecules’ charge that separates by attaching to the oppositely
charged stationary phase.

Then finally, in the elution step, the molecules of the eluent compete for the ionic
compound being purified, take their places in the stationary phase, and thus the ionic
compound is purified

For example, let us look at an anionic resin quaternary amine (-N+(CH3)3). It is a strong
anion exchanger with a commercial name of Q resin. As it is an anion exchanger (having
a positive charge on itself), it helps purify various negatively charged compounds.

Gel Filtration Chromatography

Gel filtration chromatography is also known as size exclusion chromatography and

molecular sieve chromatography. It is a separation technique, where molecules in a
mixture separate based on their sizes or molecular weights. Its most common
applications are in the separation of macromolecular complexes, such as proteins, and
polymers of industrial importance.

The resins used for gel filtration chromatography consist of spherical particles or
beads that form a porous matrix upon the column’s packing. These beads are non-
reactive and non-adsorptive. The molecules larger than the pore size of the column
elutes first since these are of different resins and particular sizes.

The molecules having sizes following the magnitudes of the pores (or smaller than
them) would penetrate them. Furthermore, they will elute so that the smallest of the
molecules would come last since they are completely entering the pores

Affinity Chromatography
It is a type of column chromatography where biomolecules disintegrate from a
mixture based on their specific interactions with the resin. It utilizes in its principle the
high specificity and interaction of two biological molecules.
 The purification and separation of the desired compound from the mixture
accomplishes when the molecule of interest (called the ligand) interacts with its target
attached to the surface of the stationary phase. It is then eluted with the help of a
solvent which competes with it for binding sites on the stationary phase.

The principle of affinity chromatography.

The most commonly used application of affinity chromatography is the separation of

a protein from a mixture. It is usually processed by using the column or resin containing
the antibodies for that protein attached. Therefore, when the protein solution passes
through the column, only the protein of interest attaches with its antibodies, and the
rest washes out. However, affinity chromatography has some disadvantages too.

Gas Chromatography

It is another type of chromatography that helps to detect and analyze compounds in

their vapor form.

The mobile phase consists of an inert carrier gas containing the sample of
examination. The stationary phase is a thin microscopic layer of a polymer or liquid
attached to the inside of a column. Experts usually term it vapor-phase chromatography
or gas-liquid partition chromatography.

High-Performance Liquid Chromatography (HPLC)

It is also a separation technique extensively utilized in analytical chemistry

and biochemistry. Also known formerly as high-pressure liquid chromatography, it
differs from regular liquid column chromatographic techniques in that it involves the
application for high pressures (50-35- bars) for its operation.

The basic principles are quite similar to traditional column chromatography. The
adsorbent initially attaches to the column walls through which the solvent containing
the compound passes with pressure. The attached compound further elutes with an
appropriate elution buffer.

HPLC strengthens a wide variety of fields by applying them. For example, it is broadly
used in the manufacturing industry to produce biological and pharmaceutical products.
Most of the recombinant proteins commercially available for research purposes are
purified using HPLC. Besides, it also helps in conventional and forensic research and
other medical purposes.

Parts of a column

A simple straight glass tube tapered at the bottom and often fitted with a top is still
commonly used The ratio of length to diameter should be greater than 20: 1; a ratio of
40 I can be taken as standard This tube is about 20-30 cm long and 2-3 cm in diameter.
This may hold 50-100 g of adsorbent and may retain several grams of the adsorbate.
The adsorbent is supported on a pig of cotton or glass wool may be employed. The
figure illustrates a simple, as well as a more sophisticated form apparatus Lang and
narrow tubes, are used for difficult separations. Columns fitted with cooling jackets are
used

when constant temperature conditions are required. Separating columns with

detachable adapters are being used. In these, the adsorbent is packed between two
sintered glass discs, and small-bore Teflon tubing is used for liquid input and output.
 ADSORBENTS

In order to obtain coloumn with good filtration properties, the absorbents should have
the following characters

• The Particle should be spherical in shape and uniform in size.

• Their mechanical stability must he great enough he prevent the formation of fine
dust which might be depended in the channels of the packing
• They should not be chemically, either with the eluting suds or with the sample
component
• They should contain small amounts of soluble components as possible.
• They should be categorically inactive and as a rule . should have neutral surface.

ADSORBTION COLOUMN

The glass wool or cotton plug is used as a support for the column. It is first kept in
position in the tube. The tube is then clamped vertically. Now the adsorbent is added in
portions so that the tube is packed uniformly with the adsorbent. Whenever any
portion is added, it is pressed from above with a flattened glass rod before the next
portion is added. This is continued till nearly two-thirds of it is filled.

SOLVENTS USED

The choice of solvents will naturally depend on the first place upon the solubility
relations of substance. The solvents generally employed possess boiling points between
40°C and 85°C. The most widely used medium is light petroleum (b.p. not above 80°C),
others are cyclohexane, carbon disulphide, benzene, chloroform, carbon tetrachloride,
methylene chloride, ethyl acetate, ethyl alcohol, acetone ether and acetic acid.

The solvents used in chromatography have three functions to perform

• They serve to introduce the mixture to the columns.

• They affect the process of development by which the zones of the chromatograph
fullest extent. When used for this purpose, the solvents are termed, developers
• They are also used to remove the required content of each zone from the
mechanically separated parts of the column, or from the column as a whole after it is
partly developed. The self for this purpose is called eluants. A good eluant must be a
liquid that can dissolve the various components of the column. While the same solvent
may serve throughout, it is the necessary to use different solvents at different stages of
the chromatographic separation

SUCCESSIVE ELUTION

For complex samples it may be necessary to use a series of solvents. For example, a
particular solvent may dissolve and elute only one group of compounds, such as
hydrocarbons, leaving the of the compounds containing other functional groups
undissolved and close to the top of the column After the group of compounds that are
soluble in this first solvent has been eluted from the column, a new solvent may be used
to dissolve and separate the second group of compounds on the column, For instance,
the first solvent used in the analysis of a complex mixture of compounds might be
biplane The latter removes paraffin from the sample. Having been removed from the
original sample, the paraffin can be further separated by means of a second
chromatographic column or by gas chromatography (or other suitable techniques). A
different solvent, such as benzene, which will elute aromatics from the sample, may
now be used on the paraffin-free original sample. After all the aromatics have been
removed, the benzene may be replaced by another solvent, such as diethyl ether, Each
change in solvent beings with the elution from the sample of a different set of organic
functional groups. This makes possible the complete separation of the sample into
compounds with common functional groups. Each set of fractions can be studied
separately by further chromatography with a different column or by IR. UV absorption
spectroscopy, or NMR. This makes possible qualitative and quantitative analysis of
increasing polarity or dielectric constant which dissolves compounds with increasing
polarity.

GRADIENT ELUTION

The abrupt change in solvents described above sometimes leads to problems,

particularly in collecting fractions near the interface between the two succeeding
mobile phases. This technique has been improved upon by wing process of gradient
elution. In this system, we solvents may be used that are completely miscible other, but
with different constants. After the sample is loaded on the top of the column, the first
solvent, which has a lower solvent strength, is used as the mobile phase After a suitable
of time a small amount of the second solvent is introduced With time, concentration in
the first solvent progressively increases until finally, the mobile phase consists entirely
of the second solvent.

DETECTORS

These are used to monitor and determine quantitatively the dissolved substances
emerging from the column. The various detectors are as follows:

(a)Optical Detectors. These are the old type of flow analyzers which are small cell
made from glass or quartz and are used for continuous photometric analysis with
visible UV light of appropriate wavelength . If the samples do not exhibit appreciable
absorption in the cited regions, regrets can be added produce colour reactions which
can be measured photometrically

(b)Differential Refractometers. Claesson has employed the refraction of the emerging

with detection. This method has been improved to a differential refractometer having a
sensitivity.
 (c)Detectors Based on Heat of Adsorption. These detectors are known as micro-
adsorption detectors. In these, the liquid emerging from a separating column is passed
through two cells, one located on top of the other while the lower cell is filled with an
adsorbent. In the centre of packing of each of the cell, the glass-covered measuring
point of a small thermometer is located. The total deflection (+ve and -ve peak) is
proportional to the concentration at least to a range of 102 .

(d)Flame Ionisation Detectors. In these detectors, there is an endless metal wire which
is passed by the column exit. The decomposition products of the substances, which are
transported by the wire are led to a flame ionisation or argon detectors.

(e)Conductivity Detectors. These are suitable for ionised substances in aqueous

solution. The effluent passed through the measuring cell of the detector which contains
two or three platinum electrodes within a Wheatstone bridge circuit and is operated by
alternating current. Other detectors used are microwave detector, ultrasonic detector,
vapour pressure detector, solvent front detector, and mass detector.